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Design of activated T cells of bispecific T cell activator and application thereof

A technology of cells and cell groups, applied in the direction of receptor/cell surface antigen/cell surface determinant, for targeting specific cell fusion, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc., can Solve the problems of neurotoxicity and CAR-T therapy not showing clinical efficacy

Pending Publication Date: 2021-03-12
普米斯生物技术(苏州)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, there are still two major problems in the use of immunotherapy to treat solid tumors: on the one hand, the severe and even fatal clinical side effects associated with CAR-T therapy are still a huge risk in the clinical application of CAR-T therapy. These side effects mainly include cytokines Release syndrome (cytokine release syndrome, CRS), macrophage activation syndrome, hemophagocytic lymphohistiocytoma, neurotoxicity, etc.
On the other hand, in the clinical treatment of solid tumors, CAR-T therapy has not yet shown its significant clinical efficacy

Method used

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  • Design of activated T cells of bispecific T cell activator and application thereof
  • Design of activated T cells of bispecific T cell activator and application thereof
  • Design of activated T cells of bispecific T cell activator and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0177] The design of embodiment 1CAB and contrast structure

[0178] 1.1 Control group CD3e-BBζ, 1 st -CAIX-BiTA, 1 st - Structure design of CAIX-CAB and CAIX-TRuC

[0179] In order to verify the anti-tumor activity of CAB-T, in a set of experiments, we first designed the first set of structures using nanobodies targeting CAIX: CD3e-BBζ (amino acid sequence is SEQ ID NO.: 7), CAIX-BiTA ( The amino acid sequence is SEQ ID NO.:8), CAIX-CAB (amino acid sequence is SEQ ID NO.:9) and CAIX-TRuC (amino acid sequence is SEQ ID NO.:10). In order to distinguish the second group of CAIX-CAB structures described later, we named the CAIX-BiTA and CAIX-CAB of this group as 1 st -CAIX-BiTA and 1 st -CAIX-CAB. Among them, 1 st -CAIX-BiTA is BiTA without label, 1 st -CAIX-CAB is the CAB structure of CD3e-BBζ and untagged BiTA, and CAIX-TRuC is the TCR-utilized 2 The comparative structure of the company's platform technology. The specific structure diagram of the above structure is as ...

Embodiment 2

[0184] Example 2 Packaging of CAB and its control structure lentiviral vector

[0185] In the present invention, we use lentivirus as a vector to prepare CAB-T cells. First, we prepared lentiviral vectors carrying genes encoding CAB and its control constructs. The specific process of lentivirus packaging is as follows:

[0186] 1) Spread 1×10 in a 10cm culture plate 7 For HEK 293T cells, add 10 mL of DMEM (Hyclone, SH30243.01) medium containing 10% FBS (Gibco, 10099-141C), mix the cells well, and culture overnight at 37 degrees;

[0187] 2) On the second day, when the HEK 293T (ATCC, CRL-3216) cell confluence reaches about 90%, replace with serum-free DMEM;

[0188] 3) Prepare the plasmid complex, the amount of each plasmid is: 8 μg plasmad DNA, 4 μg psPAX2 and 2 μg pMD2g, dissolve in 1 mL opti-MEM (Gibco, 31985-070) and add 42 μL PEI (Polysciences, 24765-2); vortex Spin and shake for 20s. After standing at room temperature for 15 minutes, gently add the mixture to the HE...

Embodiment 3

[0192] Example 3 Preparation of CAB and its control structurally engineered T cells

[0193] After the lentiviral vector carrying the CAB structure is prepared, the lentiviral vector can be used to infect immune cells to complete the preparation of CAB-T cells. The specific procedure for the preparation of CAB-T cells is as follows:

[0194] 1) Culture commercial PBMC (Sai ​​Li Bio, SLB-HP050B) cells with X-VIVO 15 (LONZA, 04-418Q) containing 5‰ human albumin (GRIFOLS, human albumin 20%), initially Cell density is 1 x 10 6 / mL;

[0195] 2) Add anti-CD3 / CD28beads (Miltenyi biotec, 130-091-441) according to the ratio of cells:Beads=3:1, and add 1000IU / mL IL-2 (Sihuan Biological, S10970016) to activate T cell expansion;

[0196] 3) After 48 hours of cell activation, an appropriate amount of virus and 12 μg / mL protamine (Sigma, P4005) were added to infect T cells;

[0197] 4) After 24 hours of lentivirus infection, the cell suspension was aspirated, and 1×10 6 Add complete f...

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PUM

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Abstract

The invention provides an immunotherapy scheme for inhibiting tumors, especially solid tumors, T cells expressing chimeric CD3e fusion protein are combined with BiTA, the chimeric CD3e fusion proteinand BiTA are combined with each other, and the functions of activating the T cells and targeting tumor cells are exerted. The invention also provides a CAB structure and a CAB editing T cell (CAB-T).BiTA secreted by the CAB-T cells can simultaneously realize activation of the CAB-T cells and activation of endogenous TCR of the non-edited T cells in tumor tissues, and the anti-tumor effect of theCAB-T and the anti-tumor effect of mobilizing the non-edited T cells are exerted. The chimeric CD3e expressed by CAB-T and BiTA cooperate with each other to exert an anti-tumor effect: activation of the chimeric CD3e depends on BiTA secreted by CAB-T, and secretion of the BiTA further stimulates the CAB-T to release more BiTA by activating the chimeric CD3e; a small amount of BiTA released by CAB-T in tumor tissues can stimulate the CAB-T to release more BiTA by activating chimeric CD3e, so that immune cell activation and anti-tumor effects of tumor tissue tendency are realized, and the safetyadvantage of CAB-T clinical application is ensured.

Description

technical field [0001] The invention belongs to the field of immunotherapy, in particular, the invention relates to the design and application of a bispecific T cell activator to activate T cells. Background technique [0002] In recent years, immune cell therapy has achieved unprecedented success in complete remission rates for hematological malignancies. In 2017, two CD19-targeting CAR-T products have been successfully launched, and were approved for the treatment of children and adolescents with acute leukemia and non-Hodgkin's lymphoma. [0003] However, there are still two major problems in the use of immunotherapy to treat solid tumors: on the one hand, the severe and even fatal clinical side effects associated with CAR-T therapy are still a huge risk in the clinical application of CAR-T therapy. These side effects mainly include cytokines Release syndrome (cytokine release syndrome, CRS), macrophage activation syndrome, hemophagocytic lymphohistiocytoma, neurotoxicit...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/29C12N5/10A61K39/00A61P35/00
CPCC07K14/7051C12N5/0636A61P35/00C12N2510/00C07K2319/02C07K2319/03A61K39/4611A61K39/4631A61K39/464454C12N5/0638A61K39/464406C07K16/2809C07K2319/33C07K2317/569C07K2317/31C07K16/30C07K16/32C07K2317/24A61K38/00A61K31/506C07K2319/00A61K2300/00A61K39/001106A61K35/17A61K2039/5156A61K2039/5158
Inventor 李志远伊刚曾竣玮刘晓林
Owner 普米斯生物技术(苏州)有限公司
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